The long-term goals of this ARCH pilot project are to develop, optimize, and assess the effectiveness of a set of molecular methods for the detection and monitoring of toxic dinoflagellates among the epiphyton and free-living communities of microorganisms in coastal waters. As a group, these toxic dinoflagellates cause a variety of public health problems, including toxic seafood and shellfish poisoning, marine animal and bird kills, respiratory distress in humans, "red tide" fish kills, etc. Such toxic microorganisms are particularly common in Florida coastal waters. The State of Florida?s Harmful Algal Bloom (HAB) Task Force has identified a critical need for the development of new technologies and approaches (particularly molecular probes) to monitor these toxins and the organisms that produce them, and how environmental variables affect their impact on public health and environmental quality. Particularly in the case of ciguatera HABs, there is a need to develop accurate and rapid methods to survey and monitor Florida waters for ciguateric dinoflagellate species and hot spots, and to test for the presence of their toxins. Specifically, this project will involve the development and field trials of assays combining genetic labeling, immunological labeling, and flow cytometry for the enumeration and cell sorting of dinoflagellate cells producing toxins of public health concern, such as ciguatoxins, maitotoxins, gambiertoxins, brevetoxins, and okadaic acid. Particular interest will focus on the detection of Gambierdiscus species causing Ciguatera Food Poisoning and on toxin-producing Prorocentrum and Gymnodinium species. Molecular assays will be developed to monitor these toxic dinoflagellates and to examine their associated bacteria, by targeting both ribosomal RNA genes and genes for polyketide synthase (PKS). Such molecular probes for Prorocentrum lima have already been developed by this group and successfully utilized with preliminary in vitro studies. The work proposed here may also serve as a model for other toxic polyketide-producing marine microorganisms. This project will take three different approaches to investigate the presence and toxic activity of these marine dinoflagellates: 1) in vitro real-time PCR and probe macroarray assays of nucleic acid extracts from epiphyton and water column microbial communities, 2) flow cytometry (FCM) assays using diagnostic light scatter and fluorescent properties of targeted dinoflagellate cells, and 3) fluorescent in situ hybridization and in situ PCR assays combined with FCM and fluorescent microscopy to assay probe-labeled cells. Multivariate analysis will be used to correlate this data to biogeochemical data generated by the investigators? ongoing Water Quality Monitoring Network, and to identify the role and effect of environmental and anthropogenic factors affecting dinoflagellate toxicity in this region.